The basis for each evolutionary development is a natural mutation rate via which the genome experiences spontaneous changes. Although the majority of these mutations do not in any way affect essential functions, a certain percentage can nevertheless have serious consequences, such as the occurrence of or predisposition to develop diseases which are passed on to the offspring (hereditary diseases). If such mutations are recessive, this does not result in any disadvantages to the individual affected as the allele or its product will still fulfil its biological role. In each new generation of a population, therefore, recessive mutations occur generally without any far-reaching change to the phenotype. Only where there is interbreeding with a second heterozygotic individual with a mutation in the same gene, are negative effects on the offspring to be expected.
Induced mutations, caused for example by the targeted use of chemicals and/or radiation, have proved extremely helpful in the genetic analysis of biological and also medical problems. If one parent is exposed to mutagenic reagents, the following F1 generation includes heterozygotic offspring who are mutation carriers, but usually do not have a changed phenotype. Only a complicated interbreeding pattern continuing into the F3 generation allows the phenotypes of these recessive mutations to be evident in homozygotic individuals. In view of the complex and time-consuming state-of-the-art processes to determine mutations of this kind, there is a need for a simpler process.
A process for detecting nonsense mutations has already been described in EP 0 872 560 whereby by means of homologous recombination a construct is produced which contains the gene to be tested downstream of a promoter and, in the same reading frame with the gene to be tested, contains a so-called reporter gene. At the expression of the reading frame, a fusion protein is formed which contains the reporter gene function. This can be detected via phenotype properties, such as resistance to antibiotics, enzymes which can be detected by appropriate processes or the ability of cells containing the construct to grow on certain culture mediums.
A disadvantage of this process however is that in an initial stage, screening must be carried out on the successful recombination of the gene fragment to be tested with the vector. Only in a second stage can the presence of the reporter function then be proven. A further problem with this process lies in the detection of heterozygotic mutations which, due to the detection of phenotypical properties, such as the enzyme activity derived from the gene product of the reporter gene, are subject to huge fluctuations, which means that in the end no reliable statement is possible. In accordance with the disclosed process, mutations are noticeable only by the absence of positive colonies. In comparison with a wild-type individual, heterozygotically mutated individuals only show a twofold difference in the number of positive colonies whereby statistically-based variations have a considerable adverse effect on the conclusions which can be drawn from the process with regard to the presence of heterozygotic mutations.